Inhibition of autophagy enhances apoptosis induced by bortezomib in AML cells

Bortezomib is a novel proteasome inhibitor, which has been successfully used to treat mantle cell lymphoma and multiple myeloma. However, the direct effects of bortezomib on acute promyelocytic leukaemia (APL) have not been fully investigated. In the present study, the WST-8 assay, western blotting, flow cytometry, monodansylcadaverine staining and transmission electron microscopy were performed. It was demonstrated that bortezomib treatment induced a time- and dose-dependent decrease in the viability of NB4 cells. Bortezomib treatment induced cell apoptosis in NB4 cells, as assessed by Annexin V/propidium iodide analysis, and the detection of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, Bax and Bcl-2 expression. Furthermore, bortezomib treatment induced autophagy in NB4 cells, as indicated by autophagosome formation, p62 degradation, LC3-I to LC3-II conversion and formation of acidic autophagic vacuoles. Notably, autophagy induced by bortezomib was initiated prior to apoptosis. Inhibition of autophagy by knocking down Beclin-1 expression increased bortezomib-induced apoptosis in NB4 cells. Therefore, the present study revealed that the combination of bortezomib and autophagy inhibition may be a potential treatment strategy for APL.     

Pharmacological doses of melatonin impede cognitive decline in tau‐related Alzheimer models,once tauopathy is initiated,by restoring the autophagic flux

Alterations in autophagy are increasingly being recognized in the pathogenesis of proteinopathies like Alzheimer's disease (AD). This study was conducted to evaluate whether melatonin treatment could provide beneficial effects in an Alzheimer model related to tauopathy by improving the autophagic flux and, thereby, prevent cognitive decline. The injection of AAV‐hTau^P301L viral vectors and treatment/injection with okadaic acid were used to achieve mouse and human ex vivo, and in vivo tau‐related models. Melatonin (10 μmol/L) impeded oxidative stress, tau hyperphosphorylation, and cell death by restoring autophagy flux in the ex vivo models. In the in vivo studies, intracerebroventricular injection of AAV‐hTau^P301L increased oxidative stress, neuroinflammation, and tau hyperphosphorylation in the hippocampus 7 days after the injection, without inducing cognitive impairment; however, when animals were maintained for 28 days, cognitive decline was apparent. Interestingly, late melatonin treatment (10 mg/kg), starting once the alterations mentioned above were established (from day 7 to day 28), reduced oxidative stress, neuroinflammation, tau hyperphosphorylation, and caspase‐3 activation; these observations correlated with restoration of the autophagy flux and memory improvement. This study highlights the importance of autophagic dysregulation in tauopathy and how administration of pharmacological doses of melatonin, once tauopathy is initiated, can restore the autophagy flux, reduce proteinopathy, and prevent cognitive decline. We therefore propose exogenous melatonin supplementation or the development of melatonin derivatives to improve autophagy flux for the treatment of proteinopathies like AD.     

基于“通肾络、益脾肾”治法研究足细胞凋亡及自噬

[目的]基于中医"通肾络、益脾肾"治法,探讨通络益肾方对体外高糖培养的小鼠肾小球足细胞自噬和凋亡蛋白SIRT1、BNIP3、P62、Bax表达的调控影响。[方法]成熟无特定病原体(SPF)级SD雄性大鼠40只,随机分为正常组、中药高、中、低剂量组各10只。按照人与大鼠体表面积折算方法计算出大鼠所需中药灌胃浓度:高剂量浓度4.76 g/mL、中剂量浓度2.38 g/mL、低剂量浓度1.19 g/m L,灌胃10 d取含药血清备用。足细胞分6组,正常组5.6 mmol/L葡萄糖+10%正常大鼠血清、高糖组30mmol/L葡萄糖+10%正常大鼠血清、通络益肾方含药血清高、中、低干预组30 mmol/L葡萄糖+10%高、中、低剂量大鼠血清、高渗组甘露醇24.5 mmol/L+10%正常大鼠血清。细胞培养48 h后收集,Hoechst33342荧光染色,观察各组足细胞凋亡状况及形态变化;流式细胞仪检测足细胞凋亡率;Western Blot检测细胞内自噬标志蛋白SIRT1、P62及促凋亡蛋白BNIP3、Bax表达水平。[结果]流式细胞仪检测结果显示通络益肾方中、低剂量组可降低高糖诱导的足细胞凋亡率(P0.01或P0.05),高剂量组不能改善凋亡情况(P0.05);Hoechst33342荧光染色观察结果也证实通络益肾方中、低剂量组可降低高糖诱导的足细胞凋亡率;蛋白印迹结果显示,与高糖组相比,通络益肾方中、低剂量组自噬标志蛋白SIRT1表达升高,自噬标志蛋白P62及促凋亡蛋白BNIP3、Bax蛋白表达下降(P0.05或P0.01),高剂量组SIRT1、BNIP3、P62、Bax蛋白表达未见明显改变(P0.05)。[结论]中剂量、低剂量通络益肾方能够启动细胞自噬减少高糖刺激下体外培养足细胞凋亡,降低细胞凋亡率及凋亡蛋白的表达。     

丹蛭降糖胶囊通过mTOR/S6K1信号通路对糖尿病肾病大鼠的干预作用研究

目的研究丹蛭降糖胶囊对糖尿病肾病大鼠的肾脏病理改变和足细胞自噬水平的影响,初步探讨其相应的作用机制。方法选取GK大鼠40只,采用醋酸脱氧皮质酮-盐皮下注射联合高脂饲料喂养诱导糖尿病肾病模型。造模成功后随机分为模型组、丹蛭降糖胶囊低剂量组[0.54 g/(kg·d)]、高剂量组[1.08 g/(kg·d)]、缬沙坦组[10 mg/(kg·d)],每组10只。另选取10只同周龄正常Wistar大鼠作为正常组。模型组和正常组给予等容积生理盐水。连续灌胃10周后检测空腹血糖(FBG)、血肌酐(SCr)、尿素氮(BUN)和尿微量蛋白(U-mAlb)。Western Blot检测肾小球p-mTOR、p-S6K1、Beclin-1、LC3和Nephrin蛋白的表达,HE染色和PAS染色后于光镜下观察肾脏病理变化,电镜下观察各组亚细胞形态结构变化。结果与正常组比较,模型组FBG、SCr、BUN、U-mAlb水平升高(P<0.01);与模型组比较,丹蛭降糖胶囊低、高剂量组和缬沙坦组U-mAlb水平降低(P<0.01)。模型组可见肾小球基底膜增厚,系膜基质沉积,足细胞损伤,各用药组均有不同程度改善,丹蛭降糖胶囊高剂量组足细胞内有较多自噬体形成。与正常组比较,模型组p-mTOR、p-S6K1的表达增高,Nephrin、Beclin-1和LC3的表达水平降低(P<0.01);与模型组比较,各用药组p-mTOR、p-S6K1的表达下降,Nephrin、Beclin-1和LC3的表达增高(P<0.01)。丹蛭降糖胶囊高剂量组和缬沙坦组Beclin1、LC3Ⅱ/LC3Ⅰ水平较丹蛭降糖胶囊低剂量组升高(P<0.05)。结论丹蛭降糖胶囊具有减轻糖尿病肾病大鼠U-mAIb、足细胞损伤及相关肾脏病理改变的作用,其机制可能与抑制mTOR/S6K1信号通路进而提高足细胞的自噬活性有关。     

自体吞噬与类固醇激素分泌调节的关系

本实验用腹腔内给予下丘脑激素或类固醇激素的方法，造成大鼠睾丸间质细胞和肾上腺皮质束状带细胞处于分泌兴奋或抑制状态，对这两种分泌类固醇激素细胞中的溶酶体和自噬小体进行了超微结构、细胞化学和形态计量研究。结果表明，在类固醇激素分泌增多时，细胞中自体吞噬活动减弱；激素分泌减少时，自体吞噬活动加强。这一结果证明了细胞内的自体吞噬活动与类固醇激素的分泌调节有密切关系，自体吞噬是溶酶体参与激素分泌调节过程的一种重要方式。     

中药单体调控自噬干预胚胎植入的研究进展

胚胎植入是生殖过程中最关键的步骤之一,植入失败的胚胎无法继续发育,是导致不孕的重要原因之一。胚胎植入的成功依赖于子宫内膜的高容受性和具有植入能力的胚胎。自噬是细胞质、细胞器和内含物被双膜囊泡吸收并运输到溶酶体进行降解和再循环的过程,是一种维持内环境稳态的方式。大量证据表明,自噬在胚胎植入的各个环节有着重要的作用。基于此探讨了自噬与子宫内膜容受性和胚胎植入能力的关系,并根据最新的研究进展,梳理了大黄素、梓醇、芍药苷、白藜芦醇、叶酸、玉米赤霉烯酮、姜黄素、汉黄芩素、槲皮素、白杨素、小檗碱、芹菜素、菲西汀、山柰酚在内的14种中药单体调控自噬干预胚胎植入的不同环节的5个机制,包括促进子宫内膜基质细胞蜕膜化、促进细胞凋亡、调节激素水平、协调炎症、促进排卵,希望对今后中药单体提高胚胎植入的成功率提供参考及思路。     

“肾虚络瘀，肾络癥瘕”病机观与肾间质纤维化中自噬不足的相关性

肾间质纤维化（RIF）是由多种病因导致慢性肾脏疾病的主要病理特征,结合中医“络病学说”“癥瘕理论”与现代医学肾脏病理特征,“肾虚络瘀,肾络癥瘕”是RIF的主要病机,正气亏虚,累及肾气,肾虚气化不利,痰热瘀毒等实邪阻滞肾络,相互胶结,形成癥瘕,积聚于肾络导致了RIF。自噬是细胞体内将受损或衰老的细胞器、变性的蛋白质等代谢产物进行降解与重吸收的过程,自噬参与了RIF发生的诸多环节,与RIF发生发展关系密切,将自噬与中医学结合研究发现,机体内生痰热瘀毒等实邪的代谢与自噬降解和重吸收功能相关,自噬在一定程度上是消除痰热瘀毒等实邪的方式,正气不足,累及肾气,肾虚气化不利,自噬功能不足,会引起痰热瘀毒等实邪累积,瘀阻肾络则易形成肾络癥瘕造成RIF,肾虚络瘀是自噬不足的基础,肾络癥瘕是自噬不足的微观病理表现,自噬不足与肾络癥瘕形成具有相同的病机演变,该文就RIF“肾虚络瘀,肾络癥瘕”病机,结合RIF-自噬病理机制,深入探讨“肾虚络瘀-自噬不足-肾络癥瘕”在RIF的相关性,全面诠释“肾虚络瘀,肾络癥瘕”病机的科学内涵,以期从中医理论阐述自噬在RIF中的作用研究奠定基础,为RIF的治疗及其机制研究提供新...     

枳实-白术调节PINK1/Parkin信号通路介导的线粒体自噬改善慢传输型便秘大鼠结肠动力障碍

目的：基于线粒体自噬及磷酸酶及张力蛋白同源物诱导的蛋白激酶1(PINK1)/帕金蛋白(Parkin)通路观察枳实、白术及其配伍对慢传输型便秘大鼠结肠动力障碍的改善作用,为临床精准用药提供理论参考。方法：将56只雄性SD大鼠按体质量随机分成正常组、模型组、自然恢复组、枳实组、白术组、枳实-白术组和莫沙必利组,每组各8只。除正常组外,采用洛哌丁胺连续14 d灌胃(3 mg·kg^-1·d^-1)构建慢传输型便秘大鼠模型。造模成功后,除模型组继续洛哌丁胺诱导外,正常组和自然恢复组采用0.9%生理盐水灌胃,枳实组(1.35 g·kg^-1·d^-1)、白术组(2.7 g·kg^-1·d^-1)、枳实-白术组(4.05 g·kg^-1·d^-1)和莫沙必利组(1.56 mg·kg^-1·d^-1)大鼠分别给予相应的药物灌胃,连续7 d。观察药物对大鼠粪便数量、粪便含水率及小肠推进率的影响;苏木素-伊...     

基于“正虚邪滞”理论探讨肺癌自噬机制及中药干预进展

自噬是一种细胞自我保护和自我更新机制,与肺癌的发生发展密切相关,有利自噬可以减缓肺癌进展,而不利自噬能够促进肺癌进展,故调节自噬水平在肺癌治疗中具有重要意义。“正虚邪滞”是王永炎院士“虚气留滞”理论的延伸,是指因肺脾肾气亏虚,引发津液代谢异常,导致痰瘀阻滞的病理过程,以肺脾肾气亏虚为本、痰瘀阻滞为标,是肺癌的关键病机。肺癌的自噬机制与“正虚邪滞”有着相互贯通之处,肺脾肾气亏虚是肺癌有利自噬减弱的关键因素,进而抑制了肿瘤细胞凋亡,并导致有害物质蓄积;痰瘀阻滞是肺癌不利自噬增强的直接因素,进而促进了正常细胞自噬性死亡,削弱了免疫细胞对肿瘤细胞的免疫抑制作用,导致肿瘤细胞增殖和迁移。正虚与邪滞相合,致使自噬状态向着不利的方向不断发展,最终导致肺癌持续进展。因此,中医干预肺癌自噬机制需以扶正抗邪为原则,补益正气以治其本,祛痰化瘀以治其标,增强有利自噬的同时抑制不利自噬,将自噬水平调节到最佳状态,从而抑制肿瘤细胞增殖和迁移,促进肺癌的缓解。通过分析现有文献可知,目前通过自噬途径治疗肺癌的中医药以中药单体为主,中医药干预肺癌自噬的作用主要集中在促进自噬激活层面,这可能是因为藉由肺脾肾气亏虚引发的有利自噬减弱是肺癌发展的根本原因。     

Beneficial Effects of a Mixture of Algae and Extra Virgin Olive Oils on the Age-Induced Alterations of Rodent Skeletal Muscle: Role of HDAC-4

Aging is associated with a progressive decline in skeletal muscle mass, strength and function (sarcopenia). We have investigated whether a mixture of algae oil (25%) and extra virgin olive oil (75%) could exert beneficial effects on sarcopenia. Young (3 months) and old (24 months) male Wistar rats were treated with vehicle or with the oil mixture (OM) (2.5 mL/kg) for 21 days. Aging decreased gastrocnemius weight, total protein, and myosin heavy chain mRNA. Treatment with the OM prevented these effects. Concomitantly, OM administration decreased the inflammatory state in muscle; it prevented the increase of pro-inflammatory interleukin-6 (IL-6) and the decrease in anti-inflammatory interleukin-10 (IL-10) in aged rats. The OM was not able to prevent aging-induced alterations in either the insulin-like growth factor I/protein kinase B (IGF-I/Akt) pathway or in the increased expression of atrogenes in the gastrocnemius. However, the OM prevented decreased autophagy activity (ratio protein 1A/1B-light chain 3 (LC3b) II/I) induced by aging and increased expression of factors related with muscle senescence such as histone deacetylase 4 (HDAC-4), myogenin, and IGF-I binding protein 5 (IGFBP-5). These data suggest that the beneficial effects of the OM on muscle can be secondary to its anti-inflammatory effect and to the normalization of HDAC-4 and myogenin levels, making this treatment an alternative therapeutic tool for sarcopenia.